Research Results in Plain English

The gluten in wheat causes illness in some individuals. (Source: USDA)

Wheat is an important staple food, but the gluten in wheat can cause illness or allergy in some people. Gluten is a term used to describe two families of proteins: glutenins and gliadins. People with Celiac Disease (CD) have painful digestive symptoms when they eat gluten. The symptoms are caused by the gliadin, not the glutenin, in wheat flour. Wheat is also a common cause of food allergies. One allergy in particular, Wheat-Dependent Exercise Induced Anaphylaxis (WDEIA) occurs when sensitive individuals ingest gluten, then engage in intense physical exercise. Like CD sufferers, people with WDEIA are reacting to gliadin, not glutenin. In fact, people with WDEIA react to a subset of gliadin proteins called omega-5 gliadins. In an effort to create a wheat flour that WDEIA-sensitive individuals can eat without risk, USDA researchers Susan Altenbach and Paul Allen used genetic engineering to reduce only the omega-5 gliadin proteins in wheat.

Altenbach and Allen attempted to reduce the omega-5 gliadin proteins in wheat grain, those that cause the WDEIA allergy, using a technique called RNAi*. The genetically engineered (GE) wheat grain from plants generated by this technique had reduced omega-5 gliadin protein, as predicted, apparently without affecting other gliadin proteins in the grain. Although not yet tested, the hope is that WDEIA susceptible individuals will be able to eat products made from this wheat without risk of allergic reaction.

As described in a previous post, RNAi was used by other researchers (see Gil-Humanes, et al., 2010) to reduce gliadin proteins in wheat. The GE wheat generated by Gil-Humanes and colleagues has reduced amounts of all gliadins, not just the omega-5 subset that causes WDEIA symptoms. Will flour made from reduced-gliadin GE wheat alleviate symptoms in people who have either CD or WDEIA? By extension, will flour made from GE wheat which is lower only in the omega-5 gliadins reduce WDEIA but not CD? Future research will answer these questions.

*RNA interference (RNAi) is a technique for preventing a gene from making protein. For more information, see this explanation on MedicineNet.

When we speak about genetically modified organisms (GMOs), we often think first about herbicide tolerant (Round-Up Ready) or insect resistant (BollGard) crops. But plants can be genetically engineered perform functions other than insect resistance and herbicide tolerance. My first post described one example: using plant genetic engineering to improve the quality of life for celiac disease patients. Bioremediation, the use of GMOs to reduce chemical contaminants in the environment, is another application of genetic engineering.

Bioremediation is the process of using live organisms to degrade toxic compounds. Phytoremediation is bioremediation using plants as the live organisms. In 2010 a research group at Nankai University published the results of their studies on phytoremediation of the herbicide, atrazine.

Atrazine is one of the most widely used herbicides in crops. Limiting weeds in the field reduces competition for water and nutrients. In corn production, for example, weed control using atrazine can lead to yield increases of 1–6%. However, atrazine is a relatively stable compound, degraded slowly in soil by microbes. It may be carcinogenic and has been associated with birth defects and endocrine disruptions. Atrazine accumulation in the soil might damage crops that are planted after atrazine treatment of fields. For these reasons, it is important to have a method to degrade atrazine in soil. One approach to increasing degradation of atrazine and other pesticides is phytoremediation.

Among the soil microbes that naturally degrade atrazine are Pseudomonas and Arthrobacter. These bacteria contain the atrazine chlorohydrolase gene (atzA), that converts atrazine to the non-toxic hydroxyatrazine. The Nankai University researchers genetically engineered (GE) plants to add the atzA gene. They tested the resulting GE plants for their ability to degrade atrazine in soil.

Non-GE plants grew as well as GE plants if the plants were grown in soil without atrazine. But in soil containing atrazine, non-GE plants were shorter and weighed less than plants genetically engineered to contain the atzA gene. Since atrazine acts by destroying chlorophyll in the leaves, the GE plants were tested for changes in chlorophyll content. When GE and non-GE plants were grown in soil containing atrazine, the chlorophyll in non-GE leaves decreased. The chlorophyll level of GE plants remained unchanged, indicating protection of the chlorophyll and therefore the plant by the introduced gene.

But will the GE plants remediate contaminated cropland? That is, can they remove atrazine from the soil, reducing damage to successive crops and reducing potential effects on human health? It’s still unclear how these plants will perform in the field. However, after growing GE plants in soil containing atrazine for 90 days, no atrazine remained in the soil. While preliminary, these data suggest that GE plants can be used for phytoremediation of an important herbicide in soil.

Genetic engineering of plants can reduce the need for chemical insecticides. This, in turn, can improve food safety, and reduce energy inputs and cost. In recent posts I described genetically engineering plants to produce their own pesticides, specifically TMOF and chitinase. I also discussed the effects that the pesticides created by those plants had on insect larvae. Savvy readers would have noticed that the effects on insects have been promising, but not stellar. It’s hard to get very enthusiastic about delayed weight gain as a measure of success!

That’s the reason the Rao research group at Università di Napoli took their project one step further and combined two biopesticides into a single plant. In a paper published in the 2010 Insect Biochemistry and Molecular Biology journal, these researchers tested the combination of plant-made TMOF and plant-made chitinase against larvae of the tobacco budworm.

Classical methods were used to breed genetically engineered (GE) plants producing TMOF with GE plants producing chitinase. The hybrid offspring produced both pesticidal proteins. Tobacco budworm(Heliothis virescens) larvae were fed leaves from the hybrid plants, or from GE plants producing either TMOF or chitinase, or from non-GE plants. The insects that were fed leaves from the TMOF-producing plants or the chitinase-producing plants developed more slowly than those fed with leaves from non-GE plants. But insects that were fed leaves from the dual-biopesticide plants developed even more slowly than the insects fed with leaves from plants producing either TMOF or chitinase alone. Most important for crop protection, approximately 75% of larvae fed on hybrid plants died. This suggests that GE plants producing both TMOF and chitinase protect themselves better against damage from insect larvae than plants producing only one of the proteins.

In a recent post, I mentioned the importance of identifying genes and proteins besides Bacillus thuringiensis (Bt) that can be used in the fight against crop pests. One such protein is chitinase.

Chitinase (pronounced ′kītən′ās) is an enzyme that breaks down the protein chitin. Chitin is an important component of insect exoskeletons and fungal cell walls. Functionally, it helps the insect or fungus retain its structure. Destroy the chitin, and the insect or fungus dies.

Scientists from the Rao laboratory at the University of Napoli hypothesized that a plant that made its own chitinase could protect itself against pests. They generated genetically engineered (GE) plants that produced chitinase, then tested the effect of the chitinase-producing GE plants on fungi and tobacco budworm larvae.

Tobacco budworm (Heliothis virescens)

The results, published in a 2008 article in Transgenic Research, showed reduced fungal growth and abnormally slow weight gain in the insect larvae after they ingested the plants. This suggests that chitinase-producing GE plants protect themselves against damage from fungi and insect larvae better than non-GE plants.

In a future article, I will report on research which suggests that combining TMOF and chitinase improves crop protection against insect pests.

One reason for genetically engineered (GE) cotton’s dramatic and rapid acceptance by farmers is its improved control of three major insect pests: tobacco budworm, cotton bollworm and pink bollworm.

Cotton and other crop plants that are genetically engineered to make Bacillus

Budworm Moth

thuringensis (Bt) proteins become resistant to damaging crop pests (see Promising Results in the Fight Against Rice Stem Borer Moths). Unfortunately, as with any pesticide, insects develop tolerance to Bt over time, so it is important to investigate other proteins that might have efficacy as biopesticides. One such protein is Trypsin Modulating Oostatic Factor (TMOF).

Tobacco Budworm Larvae

TMOF is a protein that prevents insects from synthesizing the digestive enzyme trypsin, which is critical for digestion. TMOF was first developed for use on mosquitoes. Mosquito larvae that eat TMOF die because they cannot digest their food.

TMOF can have a similar effect on crop pests. In articles published in 2002 and 2003, a research group from the University of Napoli in Italy generated GE plants that produce TMOF. The researchers then tested the effect of the TMOF-producing GE plants on insect pests. Tobacco budworm larvae that ate leaves from these plants developed at an abnormally slow rate. Later studies, which I will cover in a future post, also showed that TMOF reduced the number of larvae that survive to adulthood. Taken together, these reports show that TMOF-producing GE plants protect themselves against damage from tobacco budworm.

I also have researched the use of TMOF as a biopesticide, although not via GE plants. See one of my articles at Thompson DM, Young HP, Edens FW, Olmstead AW, LeBlanc GA, Hodgson E, Roe RM. Non-target toxicology of a new mosquito larvicide, trypsin modulating oostatic factor. Pesticide Biochemistry and Physiology 2004; 80: 131-142. DOI: 10.1016/j.pestbp.2004.06.009.

Genetic engineering promises solutions to problems of agricultural importance including plant tolerance to stress caused by drought and salinity, resistance to insect and fungal pests, and weed prevention. However, consumer concerns about genetic engineering have slowed the release of biotech crops. A genetic modification technique called oligonucleotide-directed repair (ODR) can introduce subtle improvements to plants while avoiding some of those concerns.

ODR changes a single nucleotide in a gene that is already in a plant, animal, bacteria or fungus. Remember that a gene is a piece of DNA that provides instructions to the cell. If all the DNA in the cell represents the blueprint for a house, a gene represents instructions for making a portion of the house, the front door, for instance. The instructions are written using four nucleotides, shorthanded as “A”, “C”, “G” and “T”. Change one nucleotide and you can dramatically change the instructions.

ODR is a technique for changing one nucleotide in a plant, animal, bacterium or fungus. As an example, cystic fibrosis (CF) is an inherited disease. People with CF have an error of only one nucleotide in a critical gene. The single nucleotide change leads to a build up of mucus in the lungs, often leading to patient death in their 30’s. One potential application of this technology is for gene therapy in CF sufferers.

As the name suggests, ODR is based on the ability of an oligonucleotide to cause a change in DNA. An oligonucleotide is several nucleotides put together, but shorter than a gene. The oligonucleotide differs by only one nucleotide, or one letter of the DNA code, from a gene that has always been in the cell. The organism sees the oligonucleotide and changes its gene to match it. The result is identical to the original, with the exception of one change to the instructions (gene).

C. Dong and colleagues set out to prove that ODR would work in wheat. First they introduced a defective gene into the wheat plant, using traditional genetic engineering methods. Then they tried to correct the defective gene using ODR. They used an electric pulse to introduce an oligonucleotide, into the cell. ODR made the defective gene functional, proving the utility of the technique in wheat. This technology could be used to create healthier oils in food crops or generate new industrial oils for biofuels. In wheat, it could lead to production of gluten-free flour that would be a boon to individuals with Celiac Disease.